This invention relates to neurologically-active compounds, and to methods of use thereof. In particular the invention relates co methods of enhancing cognitive activity using compounds which are antagonists of GABAC receptors. Preferred compounds for use in the methods of the invention are TPMPA and analogues thereof, and novel compounds are disclosed.
There are three major classes of GABA receptors in the central nervous system (CNS): GABAA, GABAB and GABAC receptors. The pharmacology of GABAA and GABAB receptors has been extensively investigated, but GABAC receptors have been only recognised recently, and their pharmacological potential is still unknown (Johnston, 1996b).
xcex3-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system (CNS), and activates three major subtypes of GABA receptors, the GABAA, GABAB and GABAC receptors. GABAA receptors are ligand-gated Clxe2x88x92 channels which are inhibited by the alkaloid bicuculline (Johnston, 1996a). These are heterooligomeric receptors made up of xcex1, xcex2, xcex3, and xcex4 subunits. GABAB receptors are transmembrane receptors coupled to second messenger systems and Ca2+ and K+ channels via G-proteins. These receptors are not blocked by bicuculline, but are activated by (xe2x88x92)baclofen and 3-aminopropylphosphinic acid (CGP27492) and blocked by phaclofen and saclofen (Kerr and Ong, 1995).
GABAC receptors (sometimes called GABANANB or r receptors) were first proposed when a series of conformationally restricted GABA analogues, including cis-4-aminocrotonic acid (CACA), that had bicuculline-insensitive depression actions on neuronal activity, showed no affinity for [3H]baclofen binding sites in rat cerebellar membranes (Drew et al, 1984). GABAC receptors with similar pharmacology were first found in neurons from rat retina (Feigenspan et al, 1993) and white perch retina (Qian et al, 1993). In rat retina, rod bipolar cells contain bicuculline-insensitive, baclofen-insensitive receptors that were activated by CACA (Feigenspan et al, 1993). These were detected by the co-application of GABA with 100 xcexcM bicuculline to abolish the GABAA component (Feigenspan et al, 1993). In white perch retina, rod-driven horizontal cells (H4) and not bipolar cells showed GABAC receptor-like pharmacology. Application of GABA on bipolar cells showed rapid desensitisation, while on rod-driven horizontal cells, desensitisation was not observed (Qian et al, 1993). Subsequently, GABAC receptors were found on cone-driven horizontal cells in catfish (Dong et al, 1994) and bipolar terminals in tiger salamander (Lukasiewicz et al, 1994).
The expression of mRNA from bovine retina in Xenopus oocytes showed that GABA activated two distinct GABA receptors Both receptors activated Clxe2x88x92 currents. One was mediated by GABAA receptors and was blocked by bicuculline, and the other was mediated by GABAC receptors and was insensitive to both bicuculline and baclofen (Polenzani et al, 1991). Subsequently, two cDNAs that have 30-38% sequence identity with GABAA receptor subunits were cloned from human retinal mRNA (Cutting et al, 1991; 1992). These subunits have been termed r1 and r2, and have 74% sequence identity (Cutting et al, 1991; 1992)
At least two major subtypes of GABAC receptors are now known, namely rho-1 and rho-2. As is known for other neurotransmitter receptor subtypes, different subtypes of GABAC receptors are likely so be involved in different aspects or nervous system function. As the rho-2 subunit is found in the hippocampus and neocortex, and these areas of the brain are important for memory, potent and selective agents for the rho-2 GABAC receptor are key compounds.
The species equivalents of the human r1 and r2 subunits have been cloned from rat (Enz et al, 1995). These show 88-99% homology with the respective human sequences. The use of PCR and in situ hybridisation have shown high expression of both the r1 and r2 subunits in rod bipolar cells. However, only the r2 subunit is expressed in the CNS, particularly in the hippocampus and cortex (Enz et al, 1995). Recently, a third r subunit was cloned from rat retina cDNA (Ogurusu and Shingai, 1996). This subunit exhibits 63% and 61% sequence homology to the human r1 and rat r2 sequences respectively (Ogurusu and Shingai, 1996).
Expression of human r subunits in Xenopus oocytes generates homooligomeric GABA receptors with intrinsic Clxe2x88x92 channels. These receptor ion channels are activated by GABA and CACA, but are insensitive to bicuculline, (xe2x88x92)baclofen, barbiturates and benzodiazepines. They have been shown to be sensitive to picrotoxin, and have been classified as GABAC receptors (Cutting et al, 1991; 1992; Polenzani et al, 1991; Shimada et al, 1992; Kusama et al, 1993a; 1993b; Wang et al, 1994; Bormann and Feigenspan, 1995; Johnston, 1996b).
The most potent GABAC receptor agonists known so far are trans-4-aminocrotonic acid (TACA, KD=0.6 xcexcM) and GABA (KD=1.7 xcexcM) (Woodward et al, 1993). TACA, a conformationally restricted analogue of GABA in an extended conformation, is also a GABAA receptor agonist (Johnston, 1996a). CACA, a conformationally-restricted analogue of GABA in a folded conformation, has moderate partial agonist activity at GABAC receptors (KD=74 xcexcM), and may be the most selective agonist for this receptor subtype (Johnston, 1996b).
Selective agonists and antagonists are needed to determine the physiological role of GABAC receptors and to provide more specific therapeutic agents with a lower risk of unwanted side-effects. GABA is a flexible compound, due to its rotation about the C2-C3 and C3-C4 bonds. It can exist in a range of low energy conformations (Johnston et al, 1978; Allan and Johnston, 1983). Two of these conformations have been restricted by the introduction of unsaturation in the form of a double bond at the C2-C3 position, and two compounds that represent these restricted conformations are CACA and TACA (Johnston et al, 1975). CACA and TACA have fewer degrees of rotational freedom than GABA, and can only rotate about the C3-C4 bond (Johnston et al, 1978; Allan and Johnston, 1983). CACA is a partially folded analogue of GABA. It has moderate activity at GABAC receptors expressed in Xenopus oocytes, and although its agonist activity is weak, it is to date the most selective agonist at these receptors, having minimal activity on GABAA and GABAB receptors (Johnston, 1996b). TACA is an extended analogue of GABA. It has potent agonist activity at GABAC receptors expressed in Xenopus oocytes; however, it is not selective, as it is also a potent GABAA receptor agonist (Johnston, 1996b).
Woodward et al (1993), using poly(A)+ RNA from mammalian retina expressed in Xenopus oocytes; tested many GABAA and GABAB receptor agonists and antagonists to determine a pharmacological profile for GABAC receptors. From this study, it was found that the phosphinic and methylphosphinic analogues of GABA, which are known to be potent GABAB receptor agonists, were potent antagonists at GABAC receptors.
A series of GABA analogues was tested for agonist and antagonist activity at GABAC receptors, using poly(A)+. RNA from mammalian retina injected into Xenopus oocytes. Several potent GABAC receptor antagonists were identified, including (3-aminopropyl)methylphosphinic acid (CGP35024; KB=0.8 xcexcM), 3-aminopropylphosphinic acid (CGP27492; KB=1.8 xcexcM), and 3-aminopropylphosphonic acid (3-APA, KB=10 xcexcM) (Woodward et al, 1993). These agents are not selective for GABAC receptors, as CGP35024 and CGP27492 are also very potent GABAB receptor agonists, while 3-APA is a GABAB receptor antagonist.
To date, only one specific GABAC receptor antagonist has been described. A more recently synthesised compound, 1,2,5,6-tetrahydropyridine-4-yl)methylphosphinic acid (TPMPA), does show potent and selective GABAC receptor antagonist activity (KD=2.1 xcexcM) (Murata et al, 1996; Ragozzino et al, 1996). TPMPA produces 50% inhibition or GABAC receptor activation at 2.1 xcexcM, and has the following structure: 
The effects of TPMPA on cognition are unknown.
As a result of the structure-activity relationship study and the selectivity of CACA for GABAC receptors, we have investigated the methylphosphinic acid and phosphinic acid analogues of CACA and the closely related analogue, TACA, as potential GABAC receptor antagonists. In this study, we demonstrate that the phosphinic and methylphosphinic acid derivatives of CACA and TACA, and 3-aminopropyl-n-butyl-phosphinic acid (CGP36742), an orally-active GABAB receptor antagonist, are GABAC receptor antagonists, and we have linked GABAC receptors with cognitive function. Extensive structure-activity studies were carried out on recombinant GABAC receptors from human retina expressed in frog oocytes. Among the compounds studied were a variety of compounds known to interact with GABAB receptors, provided by Ciba-Geigy AG, Basle.
The most interesting of the Ciba-Geigy compounds were a series of GABAB receptor antagonists that had been investigated in various memory and learning tests in rats and mice only one compound of the series reversed age-related deficits of old rats (Froestl, 1995b). The cognition-enhancing effects of this compound were confirmed in learning experiments in monkeys. This compound had good oral bioavailability in rats and dogs, and in healthy young and elderly male volunteers. On this basis it was selected as a development compound for the treatment of cognition deficits.
The cognition-enhancing compound, (3-aminopropyl)-n-butylphosphinic acid, code-named CGP36742, has she following structure: 
The GABAB antagonist properties of CGP36742 do not satisfactorily explain its cognition-enhancing properties, since much more potent GABAB antagonists have been described that lack these properties.
We have now surprisingly found that CGP36742 has similar potency as a GABAC antagonist to its potency as a GABAB antagonist (50% inhibition of receptor activation being found at 38 xcexcM and 62 xcexcM against GABAB and GABAC receptors respectively). None of the other potent GABAB antagonists showed activity against GABAC receptors. These findings indicate a likely role for GABAC receptor antagonism in the cognition-enhancing properties of CGP36742.
In one aspect the invention provides a method of enhancing the cognitive activity of an animal in need of such treatment, comprising the step of administering an effective amount of a compound which has GABAC receptor antagonist activity to said animal.
In a second aspect the invention provides a method of stimulating memory capacity, comprising the step of administering an effective amount of a compound which has GABAC receptor antagonist activity to an animal in need of such treatment.
The methods of the invention are suitable for the treatment of a variety of cognitive deficit conditions, dementias, and memory impairment conditions, including but not limited to those associated with Alzheimer""s disease, AIDS, and schizophrenia.
Preferably the compound has selective antagonist activity against GABAC receptors compared with GABAB receptors. More preferably, the compound has selective antagonist activity against GABAC receptors compared with GABAA receptors. Even more preferably, the compound is substantially inactive against both GABAA and GABAB receptors.
More preferably the compound comprises a phosphinic acid group, and even more preferably comprises an alkyl-substituted phosphinic acid group in which the alkyl group is of 1 to 6 carbon atoms, such as a methyl or ethyl phosphinic acid group. Most preferably the compound also comprises a double bond which imposes a conformational restriction on rotation about the bond corresponding to the C3-C4 bond of GABA. Particularly preferred compounds include, but are not limited to, conformationally-restricted analogues of CGP44530 in which rotation about the C3-C4 bond is restricted, such as TPMPA and analogues thereof.
Thus preferred compounds of the invention are represented by general formula I or general formula II, 
in which X represents hydrogen, an alkyl group optionally substituted with a halogen, or a hydroxyalkyl group, and
Y represents hydrogen, a halogen, or an alkyl, alkenyl, alkynyl or acyl group, optionally substituted wish halogen, nitrile, or NO2.
In general formula I, Y may also be an alkoxy group, optionally substituted with halogen, nitrile or NO2.
By xe2x80x9calkylxe2x80x9d is meant a straight or branched, saturated or unsaturated, substituted or unsubstituted alkyl chain of 1 to 6, preferably 1 to 4 carbon atoms, and includes alicyclic alkyl chains such as cyclopropylethyl. Alkenyl, alkynyl and acyl also refer to groups of 1-6, preferably 1-4 carbon atoms. The halogen is preferably chlorine or fluorine.
It will be clearly understood that some of the compounds which are useful for the purposes of the invention are novel, and form part of the invention. Thus in a third aspect the invention provides a compound having GABAC antagonist activity and selectivity for the rho-2 subtype of GABAC receptors of general formula II as defined above. Thus in a third aspect the invention provides a compound having GABAC antagonist activity and selectivity for the rho-2 subtype of GABAC receptors of general formula II as defined above.
In a fourth aspect, the invention provides a composition comprising a compound of general formula II, together with a pharmaceutically-acceptable carrier.
While the invention is not in any way restricted to treatment of any particular animal species, in general the animal will be a human.
The compounds may be administered at any suitable dose and by any suitable route. Oral administration is preferred because of its greater convenience and acceptability. The effective dose will depend on the nature of the condition to be treated, and the age, weight and underlying state of health of the individual to be treated, and will be at the discretion of the attending physician or veterinarian. Suitable dosage levels may readily be determined by trial and error experimentation, using methods which are well known in the art. Similarly, suitable formulations for administration by any desired route may be prepared by standard methods, for example by reference to well-known texts such Remington: The Science and Practice of Pharmacy, Volume II, 1995 (19th edition), A. R. Gennaro (Ed), Mack Publishing Company, Easton, Pa. 18042, USA., or Australian Prescription Products Guide, Volume 1, 1995 (24th edition), J Thomas (Ed), Australian Pharmaceutical Publishing Company Limited, Victoria, Australia.
Throughout the description and claims of this specification, the word xe2x80x9ccomprisexe2x80x9d and variations of the word, such as xe2x80x9ccomprisingxe2x80x9d and xe2x80x9ccomprisesxe2x80x9d, means xe2x80x9cincluding but not limited toxe2x80x9d and is not intended to exclude other additives, components, integers or steps.